The present invention relates generally to choline binding proteins, methods for isolating choline binding proteins, and the genes encoding such proteins. The invention also relates to acellular vaccines to provide protection from bacterial infection using such proteins, and to antibodies against such proteins for use in diagnosis and passive immune therapy. In particular, the choline binding proteins of the invention are useful as vaccines against pneumococcus. Where a choline binding protein demonstrates activity as an adhesion protein, it is also useful as a competitive inhibitor of bacterial adhesion, or to discover small molecule antagonists of adhesion.
Exported proteins in bacteria participate in many diverse and essential cell functions such as motility, signal transduction, macromolecular transport and assembly, and the acquisition of essential nutrients. For pathogenic bacteria, many exported proteins are virulence determinants that function as adhesions to colonize and thus infect the host or as toxins to protect the bacteria against the host""s immune system [International Patent Publication No. WO 95/06732, published Mar. 9, 1995 by Masure et al., which is specifically incorporated herein by reference in its entirety, for a review, see Hoepelman and Tuomanen, Infect. Immun., 60:1729-33 (1992)]. However, other exported proteins may not directly mediate adhesion.
Since the development of the smallpox vaccine by Jenner in the 18th century, vaccination has been an important armament in the arsenal against infectious microorganisms. Prior to the introduction of antibiotics, vaccination was the major hope for protecting populations against viral or bacterial infection. With the advent of antibiotics in the early 20th century, vaccination against bacterial infections became much less important. However, the recent insurgence of antibiotic-resistant strains of infectious bacteria has resulted in the reestablishment of the importance of anti-bacterial vaccines.
One possibility for an anti-bacterial vaccine is the use of killed or attenuated bacteria. However, there are several disadvantages of whole bacterial vaccines, including the possibility of a reversion of killed or attenuated bacteria to virulence due to incomplete killing or attenuation and the inclusion of toxic components as contaminants.
Another vaccine alternative is to immunize with the bacterial carbohydrate capsule. Presently, vaccines against Streptococcus pneumoniae employ conjugates composed of the capsules of the 23 most common serotypes of this bacterium these vaccines are ineffective in individuals most susceptible to pathological infectionxe2x80x94the young, the old, and the immune compromisedxe2x80x94because of its inability to elicit a T cell immune response. A recent study has shown that this vaccine is only 50% protective for these individuals [Shapiro et al., N. Engl. J. Med. 325:1453-60 (1991)].
An alternative to whole bacterial vaccines are acellular vaccines or subunit vaccines in which the antigen includes a bacterial surface protein. These vaccines could potentially overcome the deficiencies of whole bacterial or capsule-based vaccines.
Moreover, given the importance of exported proteins to bacterial virulence, these proteins are an important target for therapeutic intervention. Of particular importance are proteins that represent a common antigen of all strains of a particular species of bacteria for use in a vaccine that would protect against all strains of the bacteria. However, to date only a small number of exported proteins of Gram positive bacteria have been identified, and none of these represent a common antigen for a particular species of bacteria.
Recently, apparent fusion proteins containing PhoA were exported in species of Gram positive and Gram negative bacteria (Pearce and Masure, 1992, Abstr. Gen. Meet. Am. Soc. Microbiol. 92:127, abstract D-188). This abstract reports insertion of pneumococcal DNA upstream from the E. coli phoA gene lacking its signal sequence and promoter in a shuttle vector capable of expression in both E. coli and S. pneumoniae, and suggests that similar pathways for the translocation of exported proteins across the plasma membranes must be found for both species of bacteria.
In previous studies, use of random translational gene fusions (PhoA mutagenesis) to identify and alter exported proteins in Streptococcus pneumoniae provided insight into putative exported proteins [Pearce et al., Mol. Microbiol., 9:1037 (1993); International Patent Publication No. WO 95/06732, published Mar. 9, 1995; U.S. patent application Ser. No. 08/116,541, filed Sep. 1, 1993; U.S. patent application Ser. No. 08/245,511, filed May 18, 1994]. Coupling this gene fusion technology to bioactivity assays for pneumococcal adherence, the primary goal was to genetically identify and characterize immunogenic adhesion virulence determinants to eucaryotic cells that define the bacteria-host relationship and thus serve as vaccine candidates. Over 25 loci that effect adherence have been identified as determinants of virulence.
In addition, the molecular mechanism of pathogenesis caused by pneumococcus are beginning to be defined [Cundell, et al., Infect. Immun. 63:2493-2498 (1995); Wizemann, et al., Proc. Natl. Acad. Sci. USA (1996); Cundell, et al., J. Cell Biol. S18A:45 (1994); Spellerberg, et al., Mol. Microb. (1996)]. The results of these efforts shows that many bacterial components participate in a complex coordinated process to cause disease. However, it is also apparent that this strategy has produced only a few potential vaccine candidates.
Of note in the search for exported pneumococcal proteins that might be attractive targets for a vaccine is pneumococcal surface protein A (PspA) [see Yother et al., J. Bacteriol., 174:610 (1992)]. PspA has been reported to be a candidate for a S. pneumoniae vaccine as it has been found in all pneumococci to date; the purified protein can be used to elicit protective immunity in mice; and antibodies against the protein confer passive immunity in mice [Talkington et al., Microb. Pathog. 13:343-355 (1992)]. However, PspA demonstrates antigenic variability between strains in the N-terminal half of the protein, which contains the immunogenic and protection eliciting epitopes (Yother et al., supra). This protein does not represent a common antigen for all strains of S. pneumoniae, and therefore is not an optimal vaccine candidate.
Previous studies have shown that PspA, as well as one other surface exposed protein, LytA, the autolytic amidase, bind to teichoic acid (TA), an integral part of the cell wall of Streptococcus pneumoniae in a choline-dependent manner. TA contains a unique terminal phosphorylcholine moiety. PspA, a protein having a molecular weight of 84 kDa, and which is highly variable, is released from the cell surface with high choline concentration. Lyt, or autolysin, is a 36 kDa protein, which lyses the pneumococcal cell wall (self lysis), but is not released from the cell by growth in high concentrations of choline, by washing in 10% choline, or by growth in ethanolamine. Reports on choline binding proteins include those by Sanchez-Puelles et al Gene 89:69-75 (1990), Briese and Hakenback Eur. J. Biochem. 146:417-427, Yother and White J. of Bacteriol. 176:2976-2985, Sanchez-Beato et al J. of Bacteriol. 177:1098-1103, and Wren Micro. Review Mol. Microbiol. 5:797-803 (1991), which are hereby incorporated by reference in their entirety.
A variety of covalent and non-covalent mechanisms of attachment have been described for proteins decorating the surfaces of gram positive bacteria. Some streptococci and Clostridium sp. have phosphorylcholine as a unique component of the cell wall. This molecule is the terminal constituent of the teichoic acid (C polysaccharide) and lipoteichoic acid (LTA) attached to the cell wall and plasma membrane of these bacteria. A family of choline binding proteins (CPBs) have also been described which serve a variety of cellular functions. These proteins consist of an N-terminal activity domain and a repeated C-terminal signature choline binding domain that anchors these molecules to the surface of the bacteria. This motif has been identified in the C-terminal regions of a secreted glycoprotein from Clostridium acetobutylicum NCIB 88052 [Sanchez-Beato, et al., J. Bacteriol. 177:1098-1103 (1995)], toxins A and B from Clostridium difficile [Von Eichel-Streiber and Sauerborn, Gene 96:107-13 (1990); Von Eichel-Streiber et al., J. Bacteriol. 174:6707-6710 (1992)], a glucan-binding protein from Streptococcus mutans, several glycosyltransferases from Streptococcus downei and S. mutans, the murein hydrolase (LytA) from pneumococcus and pneumococcal lytic phage [Ronda et al., Eur. J. Biochem. 164:621-4 (1987); Diaz et al., J. Bacteriol. 174:5516-25 (1992); Romero et al., Microb. Lett. 108:87-92 (1993); Yother and White, J. Bacteriol. 176:2976-85 (1994)], and a surface antigen (PspA) also from pneumococcus.
S. pneumoniae is a gram positive bacteria which is a major cause of invasive infections such as sepsis and meningitis [Tuomanen et al., N. Engl. J. Med. 322:1280-1284(1995)]. The pneumococcus colonizes the nasopharyngeal epithelium and then penetrates the epithelium of the lung or nasopharynx in order to reach the vascular compartment. Such translocation would involve, of necessity, passage from an epithelial site through the underlying basement membrane/extracellular matrix and across endothelia. Pneumococci have been demonstrated to adhere to epithelia, endothelia and basement membrane in vitro and in vivo [Plotkowski et al., Am. Rev. Respir. Dis., 134 (1986); Cundell and Tuomanen, Microb Path., 17:361-374 (1994); Cundel et al., Nature, 377:435-438 (1995); van der Flier et al., Infect. Immun., 63:4317-4322 (1995).
Fibronectin is a mammalian glycoprotein present as a soluble dimer (molecular weight of 550 kDa) in body fluids such as plasma (200-700 mg/ml), cerebrospinal fluid and amniotic fluid and as a less soluble multimer in the extracellular matrix and basement membrane [Ruoslahti, Ann. Rev. Biochem., 57:375-413 (1988)]. Fibronectin has specific binding sites for a number of proteins including collagen, integrins, and two binding sites for heparin. Many microorganisms bind fibronectin, including oral streptococci and some gram negative bacteria [Westerlund and Korhonen, Mol. Microbiol., 9:687-694 (1993)]. These diverse pathogens all target the Type 1 repeats of the N-terminal heparin binding domain of fibronectin. The cognate fibronectin binding proteins demonstrate a similar amino acid sequence motif consistent with binding to the same target within fibronectin [Westerlund and Korhonen, 1993, supra]. In contrast to this pattern, Streptococcus pneumoniae was found to adhere avidly to immobilized fibronectin at the carboxyterminal heparin binding domain [van der Flier et al., Infect. Immun., 63:4317-4322, (1995)]. This domain of fibronection has a number of biological activities. It contains the major proteoglycan binding domain (Hep II) and also supports binding of the leukocyte integrin VLA-4 at two regions in the type III connecting segment (IIICS) [Wayner et al., Cell. Biol., 109:1321-1330 (1989; Guan and Hynes, Cell, 60:53-61 (1990); Mould et al., J. Biol. Chem., 266:3579-3585 (1991)]. The VLA-4 binding domain is distinct from that containing the RGD motif for binding fibronectin by VLA-5 [Pierschbacher et al., Cell, 26:259-267 (1981).
The IIICS segment is subject to alternative splicing and is absent in some soluble forms of fibronectin [Guan and Hynes, 1990, supra].
VLA-4, xcex14xcex21 (CD49d/CD29), is an integrin present on lymphocytes, monocytes, muscle cells and melanoma cells which mediates binding to VCAM-1 on endothelial cells and myoblasts and to the IIICS domain of fibronectin in the subendothelial matrix [Osborn et al., Cell, 59:1203-1211 (1989); Mould et al., J. Biol. Chem. 254:4020-4024 (1990); Shimizu et al., Immunological Reviews, 114:109-143 (1990); Rosen et al., Cell, 69:1107-1119 (1992)]. These interactions are important during infiltration of mononuclear cells to sites of inflammation, metastasis of melanoma cells and in myogenesis [McCarthy et al., J. Cell. Biol. 102:179-188 (1986); Osborn et al., 1898, supra; Shimuzu et al., 1990, supra; Rosen et al., 1992, supra]. VLA-4 targets a 25 amino acid region (CS1) with the IIICS domain of fibronectin, an interaction which can be blocked by the tripeptide Leu-Asp-Val [Guan and Hynes, 1990, supra; Komoriya et al., J. Biol. Chem. 266:15075-15079 (1991); Mould et al., 1991, supra]. An homologous motif IDSP is present in the VLA-4 binding sites in VCAM-1 domains I and IV [Clements et al., J. Cell. Sci., 107:2127-2135 (1994)]. The binding sites on VLA-4 for VCAM-1 and fibronection have been suggested to be distinct but overlapping [Elices et al., Cell, 50:577-584 (1990); Pulido et al., J. Biol. Chem., 266:10241-10245 (1991); Vonderheide and Springer, J. Exp. Med., 175:1433-1442 (1992); Makarem, J. Biol. Chem. 269:4005-4011 (1994)]. The ability of pneumococci to target the HepII region of fibronectin raised the possibility that these bacteria recognized the region common between HepII and VCAM-1 and that the binding was mediated by a bacterial version of VLA-4. Such interactions could promote passage of pneumococci across the basement membrane. The importance of VLA-4-VCAM-1 interactions for leukocyte trafficking to brain also suggested a role for pneumococcal transmigration into the central nervous system in meningitis.
Therefore, in view of the aforementioned deficiencies attendant with prior art methods of vaccinating against bacterial pathogens, it should be apparent that there still exists a need in the art for identifying protein antigens suitable for use as subunit vaccines, and for use in inducing antibodies suitable for use in passive immunization.
The citation of any reference herein should not be construed as an admission that such reference is available as prior art to the invention.
In accordance with the present invention, bacterial surface antigens are provided which are suitable for use in immunizing animals against bacterial infection. More particularly, novel choline binding proteins from streptococci, preferably pneumococci, are provided.
In a further embodiment, a method is provided for isolating and identifying choline binding proteins, and the genes encoding them.
In its broadest aspect, the present invention extends to streptococcal surface antigens, generally referred to herein as choline binding proteins, having the following characteristics:
a) binding to choline; and
b) eluting from a choline affinity chromatographic column in the presence of 10%, preferably at least 10%, choline in Dubelcco""s phosphate buffered saline (DPBS);
with the proviso that the bacterial surface antigen of the present invention is not PspA or autolysin (LytA). In a preferred aspect, the choline binding protein of the invention has one or more of a characteristic selected from the group consisting of:
c) inhibiting adherence of the bacteria to host cells;
d) being reactive with sera from patients infected or recovering from infection with the bacteria;
e) being reactive with rabbit antisera generated against purified choline binding proteins isolated from a choline affinity column by elution in 10% choline, DPBS; and
f) labeled by fluorescein isothiocyanate (FITC) without requiring bacterial lysis (i.e., in intact bacteria).
In a specific example, the bacterial surface antigen is isolated from pneumococcus.
In a still further aspect, the present invention extends to vaccines based on the choline binding proteins.
In a particular embodiment, the present invention relates to all members of the herein disclosed family of bacterial surface antigens which bind choline, with the proviso that this group does not include PspA or LytA.
In a preferred embodiment, the invention provides a choline binding protein with a high degree of sequence similarity to enolase, particularly B. subtilis enolase.
The present invention also relates to an isolated nucleic acids, such as recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a bacterial choline binding protein (CBP) of the invention. Preferably, the nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the CBP has a nucleotide sequence or is complementary to a DNA sequence or fragment thereof which codes on expression for an amino acid having a sequence as follows:
Almost Full Length CBP50 (SEQ ID NO: 19)
Partial CBP112 (SEQ ID NO:21)
In a specific embodiment a nucleic acid of the invention encodes at least a portion (an internal fragment) of the choline binding protein CBP50, preferably having a nucleotide sequence depicted in SEQ ID NO:18; or a portion of the 5xe2x80x2 region of the gene encoding binding protein CBP112, preferably having a nucleotide sequence depicted in SEQ ID NO:20.
The DNA sequences encoding the CBPs of the present invention, or portions thereof, may be prepared as probes to screen for complementary sequences and genomic clones in the same or alternate species. The present invention extends to probes so prepared that may be provided for screening. For example, the probes may be prepared with a variety of known vectors, such as the phage xcex vector. Such probes are useful for diagnosis, e.g., to confirm a species or strain of Gram positive bacterial infection, as well as for cloning cDNA or genes encoding CBPs.
The present invention also includes CBPs having the activities noted herein, and that display the amino acid sequences set forth and described above and selected from SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 19, and 21.
In a further embodiment of the invention, the full DNA sequence of the recombinant DNA molecule or cloned gene so determined may be operatively linked to an expression control sequence which may be introduced into an appropriate host. The invention accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding the present CBP(s), and more particularly, the DNA sequences or fragments thereof determined from the sequences set forth above and in SEQ ID NOS:18 and 20.
According to other preferred features of certain preferred embodiments of the present invention, a recombinant expression system is provided to produce biologically active CBPs or immunologically reactive portions thereof.
The concept of the bacterial surface antigens contemplates that specific factors exist for correspondingly specific binding proteins, such as CBPs and the like, as described earlier. Accordingly, the exact structure of each CBP will understandably vary so as to achieve this choline-binding and activity specificity. It is this specificity and the direct involvement of the CBP in the adherence of the bacteria, that offers the promise of a broad spectrum of diagnostic and therapeutic utilities.
The present invention naturally contemplates several means for preparation of the CBPs, including as illustrated herein known recombinant techniques, and the invention is accordingly intended to cover such synthetic preparations within its scope. The isolation of the DNA and amino acid sequences disclosed herein facilitates the reproduction of the CBPs by such recombinant techniques, and accordingly, the invention extends to expression vectors prepared from the disclosed DNA sequences for expression in host systems by recombinant DNA techniques, and to the resulting transformed hosts.
A particular advantage of the present invention is that it provides for preparation of sufficient quantities of CBPs for commercialization of anti-CBP vaccines. A further advantage is that the invention provides for preparation of multi-component vaccines containing two or more CBPs, thus broadening and increasing the potential effectiveness of the vaccine. In its primary aspect, the invention contemplates utilizing the CBPs of the invention, either separately or in combinations of two or more, in vaccines for protection against pneumococcal infection. Preferably, a vaccine comprising two or more CBPs further comprises PspA, LytA, or both. According to the invention, CBPs may be prepared recombinantly. Alternatively, CBPs may be obtained from bacterial cultures, e.g., using the purification methods described and exemplified herein. In a specific embodiment, a mixture of CBPs from pneumococcus are obtained, e.g., by choline affinity chromatography, and are used directly without further purification to immunize an animal and elicit protective antibodies. Such a mixture of choline affinity purified proteins may be obtained from bacteria that express PspA or that lack PspA expression (i.e., PspAxe2x88x92 bacteria as exemplified herein).
In another aspect, the genes (e.g., cDNA) encoding one or more CBPs of the invention are engineered in a transgenic vector for expression in a mammalia host in vitro, as a nucleic acid-based vaccine.
In yet another embodiment, the CBPs of the invention are used to generate antibodies for passive immunization, diagnostics, or screening. In a specific example, infra, passive immunization prevents death from pneumococcal infection in a murine model.
The diagnostic utility of the present invention extends to the use of binding partners, notably antibodies, to the present CBPs in assays to screen for bacterial infection.
Antibodies against the CBP(s) include naturally raised and recombinantly prepared antibodies. Such antibodies could be used to screen expression libraries to obtain the gene or genes that encode the CBP(s). These may include both polyclonal and monoclonal antibodies prepared by known genetic techniques, as well as bi-specific (chimeric) antibodies, and antibodies including other functionalities suiting them for additional diagnostic use conjunctive with their capability of modulating bacterial adherence.
Thus, the CBPs, their analogs and/or analogs, and antibodies that may be raised thereto, are capable of use in connection with various diagnostic techniques, including immunoassays, such as a radioimmunoassay, using for example, an antibody to the CBP that has been labeled by either radioactive addition, or radioiodination.
In an immunoassay, a control quantity of the antagonists or antibodies thereto, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a cellular sample. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached.
In the instance where a radioactive label, such as the isotopes 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 186Re are used, known currently available counting procedures may be utilized. In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.
The present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of the bacterial infection, or to identify drugs or other agents that may mimic or block such infection. The system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the CBPs, their agonists and/or antagonists, and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand, capable either of binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s).
In particular, the proteins of CBPs whose sequences are presented in SEQ ID NOS:1-10, 19 and 20 herein, their antibodies, agonists, antagonists, or active fragments thereof, could be prepared in pharmaceutical formulations for administration in instances wherein antibiotic therapy is appropriate, such as to treat or prevent bacterial infection. Such free proteins could compete with bacterial CBP function, thus interfering with bacterial pathological activity such as adhesion.
In a preferred aspect, the invention provides a method and associated compositions for treating infection with a bacterium that expresses a streptococcal choline binding protein comprising administering pulmonarily an adhesion inhibitory agent selected from the group consisting of a choline binding protein having the following characteristics:
choline-binding activity; and
elution from a chromatographic column in the presence of 10% choline;
with the proviso that the streptococcal choline binding protein is not PspA or autolysin (LytA), an antibody to the choline binding protein, an enolase, a hindered cationic small molecule, the peptide WQPPRARI (SEQ ID NO:11), and an antibody specific for an epitope having the amino acid sequence WQPPRARI (SEQ ID NO:11). Preferably, the hindered cationic small molecule is selected from the group consisting of lysine, choline, and arginine. In a further embodiment, the adhesion inhibitory agent is administered with another drug, such as an antibiotic, a steroid, a non-steroidal anti-inflammatory drug, etc.
Accordingly, it is a principal object of the present invention to provide a CBP and its subunits in purified form.
It is a further object of the present invention to provide antibodies to the CBP and its subunits, and methods for their preparation, including recombinant means.
It is a further object of the present invention to provide a method for detecting the presence of the CBP and its subunits in mammals in which invasive, spontaneous, or idiopathic pathological states are suspected to be present.
It is a still further object of the present invention to provide method for the treatment of mammals to control the amount or activity of the bacteria, the CBP or subunits thereof, so as to alter the adverse consequences of such presence or activity, or where beneficial, to enhance such activity.
It is a still further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of the bacteria or its subunits, so as to treat or avert the adverse consequences of invasive, spontaneous, or idiopathic pathological states.
It is a still further object of the present invention to provide pharmaceutical compositions for use in therapeutic methods which comprise or are based upon the CBP, its subunits, or their binding partner(s).
Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing description which proceeds with reference to the following illustrative drawings and Detailed Description of the Invention.